Compositions and methods for controlling head blight disease

ABSTRACT

Compositions comprising microbiological strains and cultures and methods of use thereof are provided herein. Certain strains, cultures, and compositions thereof are useful for the control of head blight disease, for example, of various crop plants. Biological control compositions, and methods of use thereof to prevent, inhibit or treat the development of plant pathogens or disease and for preserving plant yield, are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/511,467, filed on Jul. 25, 2011, the entire contents of which is herein incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

The material in the accompanying Sequence Listing is hereby incorporated by reference in its entirety. The accompanying file, named “SGI1520-1_ST25.txt”, was created on Jul. 24, 2012 and is 52 Kb. The file can be accessed using Microsoft Word on a computer that uses Window OS.

FIELD OF THE INVENTION

The present invention relates to the biological control of phytopathogenic diseases. Specifically, it relates to compositions and methods useful for controlling head blight disease in cereal plants, such as wheat and barley.

BACKGROUND OF THE INVENTION

Head blight, also known as head scab, pink mold, whiteheads and tombstone scab, is a devastating disease afflicting wheat, barley, and several other cereal crops worldwide, particularly in USA, Europe and China. This disease can reach epidemic levels and cause extensive damage to grains, especially wheat and barley in humid and semi-humid cereal growing areas of the world, including India, Russia, France, Germany, and the United Kingdom. Particularly, wheat scab or head blight is one of the more damaging diseases of wheat in the United States. Nationwide, this disease has caused the wheat industry millions of dollars in yield losses. In the Midwest and High Plains, wheat scab is the major obstacle to wheat production in recent years. Head blight in addition to attacking wheat also attacks and reproduces on barley, oats, corn, and many other cereals.

This serious plant disease can be caused by several phytopathogens, but primarily by several species in the fungal genus Fusarium. For example, causal agents of the head blight disease in wheat include a number of different Fusarium species, e.g. F. culmorum, F. graminearum (teleomorph, Gibberella zeae), F. avenaceum (teleomorph, G. avenacea), F. poae, as well as by non-Fusarium pathogens such as Microdochium nivale (teleomorph, Monographella nivalis), and Microdochium majus. In the United States, Europe and most agronomically important areas of the world, the predominant causative agent of head blight is Fusarium graminearum (teleomorph, Gibberella zeae sensu strict).

These pathogens typically survive on plant debris. They invade and damage the spikelets of the grain head during flowering, thus preventing or partially impeding the development of grain in the grain head. As a result, the invading scab pathogen can either kill part of the grain head or the entire grain head. Some infected seeds are low in vigor and often fail to germinate. Infected seeds that germinate often die early in the seedling stage due to crown rot or root rot, causing poor stands in the following crop plant. Healthy seedlings can also become infected at emergence. In addition to poor, unthrifty stand, yield losses due to pathogen infestation can be quite high if conditions are favorable for development of the disease.

The fungal pathogens of Fusarium genus spread across grain cultivating areas all over the world, and are known to cause severe damage, particularly in areas with high rainfall between flowering and grain filling. When Fusarium graminearum is the causative agent, this disease is of primary concern because it does not only reduce the commercial values of the contaminated grains, in addition to yield losses, but because Fusarium infection can also lead to the accumulation of trichothecene mycotoxins in the grains thus threatening the health of human and livestock. Trichothecenes are major mycotoxin contaminants of cereals worldwide, causing feed refusal, vomiting, diarrhea and weight loss in non-ruminant animals and posing a health threat to other animals and humans when exposure levels are high. This threat has been exacerbated by the recent shift in the F. graminearum strains in the United States towards greater toxin production and vigor. Most frequently found mycotoxins are deoxynivalenol (DON, also known as vomitoxin) and zearalenone (ZEA). Deoxynivalenol in particular is a very dangerous toxin, causing gastrointestinal disorders accompanied by hemorrhagic conditions and the like in humans and animals that eat infected grains, leading to death in some cases. Since deoxynivalenol is generally stable against changes in pH and high temperature, detoxification can be very difficult. Therefore, grains contaminated beyond a certain level cannot be used in any form of brewing, processing, or livestock feed, and thus often need to be disposed of.

To date, various strategies have been deployed for controlling head blight in crop plants. Promising options include chemical measures, the development of resistant crop cultivars, and traditional practices of crop rotation and tillage of fields. Among these options, chemical pesticides can be somewhat effective in reducing head blight infestation, but residue concerns regarding the use of fungicides late in crop development, typically at flowering stages only a few weeks before harvest, lessen their attractiveness. Advances in developing resistant cultivars using traditional breeding and genetic engineering represent another disease control alternative are also occurring. Reported examples of genetic engineering advances include altering the production of a plant hormone or manipulating the plant hormone signaling pathway. In recent years, considerable advances in the area of traditional breeding have been made in understanding the genetic basis of resistance to head blight disease and a number of genes and quantitative trait loci (QTL) conferring resistance have been reported. However, progress in improving crop resistance to head blight disease has been slow, largely because of the difficulty of studying this disease. In fact, relatively little is currently understood about the mechanisms involved in resistance or susceptibility. In addition, the genetic diversity of Fusarium species, which are the predominant causative agents of the disease, often raises concerns regarding how durable the efficacy of chemical fungicides and resistant cultivars will be. As a result, practically all wheat cultivars currently in large-scale production remain vulnerable to infection.

In addition, although some success in controlling head blight disease can be expected by traditional practices such as plowing fields to bury crop residues infested with causative agent, e. g. F. graminearum, after harvest, conventional tillage of fields after harvesting is not compatible with the soil conservation practice of minimum tillage. Considering the potential of long distance inoculum dispersal and the diverse crops that can act as alternative hosts of the pathogens, crop rotation is often an untenable solution. In addition to the problem of pesticide residues in the environment, reports of pesticide resistance and instances of DON content increases in grain can also be concerns with their use. Further, costs and increasing concerns in the public and private sectors over pesticide residues in the environment and food product safety render this disease control alternative less attractive, and have led to requests for crop cultivation using as little pesticides as possible.

In summary, despite considerable advances in developing techniques to control head blight, reducing the impact of this devastating disease on grain production and quality remains an intractable problem. Therefore, identification and development of new head blight controlling techniques is essential in improving the production and quality of many cereal crops. These problems require urgent solution not only in United States, but also across the globe, including the Asia and Europe.

Biological control of head blight disease has attracted considerable interest since the mid 1990′s. Biological control agents (BCAs), though currently very limited in number, could be an environmentally acceptable method for substantially decreasing the level of disease incited by pathogens such as Fusarium. Public acceptance, compatibility with other disease management measures, and durability are among favorable factors in support of developing strategies for biologically controlling head blight disease. Biological control agents could play an important role in organic cereal production. In conventional production, such agents may extend protection of spikes past the flowering stage after chemical fungicides can no longer be applied. To date, significant advances in the area of biological control have been achieved. For example, certain strains of spore-producing bacteria (such as Bacillus and Pseudomonas species) and yeasts (such as Cryptococcus species) show some promise for the control of head blight disease and the reduction of mycotoxin contamination. However, despite these and other advances, the need remains for improved microorganisms for use in the biological control of head blight disease. Although BCAs have become a more acceptable solution for plant pathogens and BCA products have been marketed to a greater extent than ever before, to date there have been few attempts to develop strategies and antagonistic microorganism for biologically controlling head blight disease. Furthermore, the life cycle of Fusarium spp. and other causative agents of head blight disease suggest that the pathogens can potentially be susceptible to biocontrol techniques using antagonistic microorganisms at different developmental stages. Thus, there is a need to identify new biological control agents, preferably with different modes of actions, as well as biocontrol methods that can help effectively prevent or suppress the development of head blight disease.

SUMMARY OF THE INVENTION

Compositions comprising microbiological strains and cultures are provided herein. Certain strains, cultures, and compositions thereof are useful for the control of head blight disease, for example, of various crop plants including wheat and other cereal plants. Biological control compositions, and methods of use thereof to prevent, inhibit or treat the development of plant pathogens or disease and for preserving plant yield, are also provided. Also provided are methods for the use of such compositions as biological control agents in combination with other agriculturally effective compounds or compositions for controlling harmful plant pathogens.

In one aspect, the present invention provides isolated microbial strains having suppressive activity against head blight disease. The microbial strains in accordance to this aspect of the present invention are selected from the group consisting of the genera Microbacterium, Bacillus, Mycosphaerella, and Variovorax. In some preferred embodiments, the microbial strains are selected from the group consisting of Mycosphaerella sp. strain SGI-010-H11 (deposited as NRRL 50471), Microbacterium sp. strain SGI-014-006 (deposited as NRRL B-50470), Microbacterium sp. strain SGI-005-G08 (deposited as NRRL__-______), Variovorax sp. strain SGI-014-G01 (deposited as NRRL B-50469), Bacillus sp. strain SGI-015-F03 (deposited as NRRL B-50760), Bacillus sp. strain SGI-015-H06 (deposited as NRRL B-50761), and pesticidally active variants of any thereof. The microbial strain in accordance to some other preferred embodiments can comprise a DNA sequence that exhibits at least 85% sequence identity to any one of the nucleotide sequences in the Sequence Listing. Further provided are biologically pure cultures and enriched cultures of the microbial strains disclosed herein.

Also provided in another aspect of the present invention are compositions that comprise a microbial strain of the invention or a culture thereof, and an agriculturally effective amount of a compound or composition selected from the group consisting of an acaricide, a bactericide, a fungicide, an insecticide, a microbicide, a nematicide, a pesticide, and a fertilizer. The compositions in some embodiments of this aspect may be prepared as a formulation selected from the group consisting of an emulsion, a colloid, a dust, a granule, a pellet, a powder, a spray, an emulsion, and a solution. In some other embodiments, the compositions may be provided with a carrier. In some preferred embodiment, the carrier is an agriculturally acceptable carrier. In some particularly preferred embodiment, the carrier is a plant seed. In other preferred embodiments, the composition is a seed coating formulation. Further provided in the present disclosure are seeds that are coated with a composition according to the present invention.

In another aspect of the present invention, there are provided methods for preventing, inhibiting or treating the development of a plant pathogen. The methods involve growing a microbial strain of the invention or a culture thereof in a growth medium or soil of a host plant prior to or concurrent with host plant growth in the growth medium or soil. In some preferred embodiments of this aspect, the plant pathogen causes head blight disease. In some particularly preferred embodiments, the plant pathogen is Fusarium graminearum.

In yet another aspect of the present invention, methods are provided for preventing, inhibiting or treating the development of head blight disease of a plant. The methods involve applying to the plant, or to the plant's surroundings, an effective amount of a microbial strain of the invention or a culture thereof. In one embodiment, such head blight disease is caused by the fungus Fusarium graminearum. In some embodiments of this aspect, the microbial strain or a culture thereof is applied to soil, a seed, a root, a flower, a leaf, a portion of the plant, or the whole plant. In a preferred embodiment, the plant is susceptible to Fusarium graminearum. In some other preferred embodiments, the plant is wheat plant, a corn plant, a barley plant, or an oat plant. In another preferred embodiment, the microbial strain of the present invention or a culture thereof is established as an endophyte on the plant.

Another further aspect of the invention provides non-naturally occurring plants. The non-naturally occurring plants are artificially infected with a microbial strain of the invention or a culture thereof Further provided in some preferred embodiments of this aspect are seed, reproductive tissue, vegetative tissue, plant parts, and progeny of the non-naturally occurring plants.

Yet another aspect of the invention provides a method for preparing an agricultural composition. The method involves inoculating the microbial strain according to the present invention or a culture thereof into or onto a substratum and allowing it to grow at a temperature of 1-37° C. until obtaining a number of cells or spores of at least 10²-10³ per milliliter or per gram.

These and other objects and features of the invention will become more fully apparent from the following detailed description of the invention and the claims.

DETAILED DESCRIPTION OF THE INVENTION Some Definitions

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof.

The expressions “antagonistic microorganism” and “microbial antagonist” are used herein interchangeably in reference to a microorganism is intended to mean that the subject strain exhibits a degree of inhibition of head blight disease exceeding, at a statistically significant level, that of an untreated control.

Antibiotic: the term “antibiotic”, as used herein, refers to any substance that is able to kill or inhibit the growth of a microorganism. Antibiotics may be produced by any one or more of the following: 1) a microorganism, 2) a synthetic process, or 3) a semisynthetic process. An antibiotic may be a microorganism that secretes a volatile organic compound. Furthermore, an antibiotic may be a volatile organic compound secreted by a microorganism.

Bactericidal: the term “bactericidal”, as used herein, refers to the ability of a composition or substance to increase mortality or inhibit the growth rate of bacteria. Inhibition of the growth rate of bacteria can be commonly quantified as the reduction of viable bacterial cells over time.

Biological control: the term “biological control” and its abbreviated form “biocontrol”, as used herein, is defined as control of a pathogen or insect or any other undesirable organism by the use of at least a second organism other than man. An example of a known mechanism of biological control is the use of microorganisms that control root rot by out-competing fungi for space on the surface of the root, or microorganisms that either inhibit the growth of or kill the pathogen. The “host plant” in the context of biological control is the plant that is susceptible to disease caused by the pathogen. In the context of isolation of an organism, such as a fungal species, from its natural environment, the “host plant” is a plant that supports the growth of the fungus, for example, a plant of a species the fungus is an endophyte of.

The term “cereal” as used herein is intended to refer to any cereal species that can be susceptible to head blight disease. Cereals reported to be susceptible include wheat, barley, oats, and triticale, though wheat and barley are the two crops in which this disease presents a significant economic problem.

An “effective amount” refers to an amount sufficient to affect beneficial or desired results. In terms of disease management, treatment, inhibition or protection, an effective amount is that amount sufficient to suppress, stabilize, reverse, slow or delay progression of the target infection or disease states. As such, the expression “an effective amount” is used herein in reference to that quantity of antagonist treatment which is necessary to obtain a reduction in the level of pathogen development and/or in the level of pathogenic disease relative to that occurring in an untreated control. Typically, an effective amount of a given antagonist treatment provides a reduction of at least 20%; or more typically, between 30 to 40%; more typically, between 50-60%; even more typically, between 70 to 80%; and even more typically, between 90 to 95%, relative to the level of disease and/or the level of pathogen development occurring in an untreated control under suitable conditions of treatment. An effective amount can be administered in one or more administrations. The actual rate of application of a liquid formulation will usually vary from a minimum of about 1×10³ to about 1×10¹⁰ viable cells/mL and preferably from about 1×10⁶ to about 5×10⁹ viable cells/mL. Under most conditions, the antagonistic microbial strains of the invention described in the Examples below, would be optimally effective at application rates in the range of about 1×10⁶ to 1×10⁹ viable cells/mL, assuming a mode of application which would achieve substantially uniform contact of at least about 50% of the plant tissues. If the antagonists are applied as a solid formulation, the rate of application should be controlled to result in a comparable number of viable cells per unit area of plant tissue surface as obtained by the aforementioned rates of liquid treatment. Typically, the biological control agents of the present invention are biologically effective when delivered at a concentration in excess of 10⁶ CFU/g (colony forming units per gram), preferably in excess of 10⁷ CFU/g, more preferably 10⁸ CFU/g, and most preferably at 10⁹ CFU/g.

Further, the expression “effective microbial antagonist” used herein in reference to a microorganism is intended to mean that the subject microbial strain exhibits a degree of inhibition of head blight disease exceeding, at a statistically significant level, that of an untreated control. Typically, an effective microbial antagonist has the ability to effect a reduction of at least 20%; or more typically, between 30 to 40%; more typically, between 50-60%; even more typically, between 70 to 80%; and even more typically, between 90 to 95%, relative to the level of disease and/or the level of pathogen development occurring in an untreated control under suitable conditions of treatment.

Composition: A “composition” is intended to mean a combination of active agent and at least another compound, carrier or composition, inert (for example, a detectable agent or label or liquid carrier) or active, such as a pesticide.

Isolated microbial strain, isolated culture, biologically pure culture, and enriched culture: As used herein, the term “isolated” as applied to a microorganism (e.g., bacterium or microfungus) refers to a microorganism which has been removed and/or purified from an environment in which it naturally occurs. As such, an “isolated strain”of a microbe as used herein is a strain that has been removed and/or purified from its natural milieu. Thus, an “isolated microorganism” does not include one residing in an environment in which it naturally occurs. Further, the term “isolated” does not necessarily reflect the extent to which the microbe has been purified. A “substantially pure culture” of the strain of microbe refers to a culture which contains substantially no other microbes than the desired strain or strains of microbe. In other words, a substantially pure culture of a strain of microbe is substantially free of other contaminants, which can include microbial contaminants as well as undesirable chemical contaminants. Further, as used herein, a “biologically pure” strain is intended to mean the strain separated from materials with which it is normally associated in nature. Note that a strain associated with other strains, or with compounds or materials that it is not normally found with in nature, is still defined as “biologically pure.” A monoculture of a particular strain is, of course, “biologically pure.” As used herein, the term “enriched culture” of an isolated microbial strain refers to a microbial culture that contains more than 50%, 60%, 70%, 80%, 90%, or 95% of the isolated strain.

As used herein, an “endophyte” is an endosymbiont that lives within a plant for at least part of its life without causing apparent disease. Endophytes may be transmitted either vertically (directly from parent to offspring) or horizontally (from individual to unrelated individual). Vertically-transmitted fungal endophytes are typically asexual and transmit from the maternal plant to offspring via fungal hyphae penetrating the host's seeds. Bacterial endophytes can also be transferred vertically from seeds to seedlings (Ferreira et al., 2008). Conversely, horizontally-transmitted endophytes are typically sexual, and transmit via spores that can be spread by wind and/or insect vectors. Endophytes of crop plants have been receiving considerable attention with respect to their ability to control both disease and insect infestation, as well as promoting plant growth.

Functionally comparable protein: The phrase “functionally comparable protein” as used herein describes those proteins that have at least one characteristic in common. Such characteristics include sequence similarity, biochemical activity, transcriptional pattern similarity and phenotypic activity. Typically, the functionally comparable proteins share some sequence similarity or at least one biochemical. Within this definition, homologs, orthologs, paralogs and analogs are considered to be functionally comparable. In addition, functionally comparable proteins generally share at least one biochemical and/or phenotypic activity. Functionally comparable proteins will give rise to the same characteristic to a similar, but not necessarily the same, degree. Typically, functionally comparable proteins give the same characteristics where the quantitative measurement due to one of the comparables is at least 20% of the other; more typically, between 30 to 40%; more typically, between 50-60%; even more typically, between 70 to 80%; even more typically, between 90 to 95%; even more typically, between 98 to 100% of the other.

Fungicidal: As used herein, “fungicidal” refers to the ability of a composition or substance to decrease the rate of growth of fungi or to increase the mortality of fungi.

Fusarium fungus: For purposes of this invention it is understood that the use of term Fusarium fungus is intended to include both the sexual (teleomorphic) stage of this organism and also the asexual (anamorphic) stage, also referred to as the perfect and imperfect fungal stages, respectively. For example, the anamorphic stage of Fusarium graminearum is Gibberella zeae, a causative agent of head blight disease. This disease typically occurs when the flower or seed head becomes inoculated with conidia produced by the imperfect form or ascospores produced by the perfect form of this fungus.

Mutant: As used herein, the term “mutant” or “variant” in reference to a microorganism refers to a modification of the parental strain in which the desired biological activity is similar to that expressed by the parental strain. For example, in the case of Microbacterium the “parental strain” is defined herein as the original Microbacterium strain before mutagenesis. Mutants or variants may occur in nature without the intervention of man. They also are obtainable by treatment with or by a variety of methods and compositions known to those of skill in the art. For example, a parental strain may be treated with a chemical such as N-methyl-N′-nitro-N-nitrosoguanidine, ethylmethanesulfone, or by irradiation using gamma, X-ray, or UV-irradiation, or by other means well known to those practiced in the art.

Nematicidal: The term “nematicidal”, as used herein, refers to the ability of a substance or composition to increase mortality or inhibit the growth rate of nematodes.

Pathogen: The term “pathogen” as used herein refers to an organism such as an alga, an arachnid, a bacterium, a fungus, an insect, a nematode, a parasitic plant, yeast, a protozoan, or a virus capable of producing a disease in a plant or animal. The term “phytopathogen” as used herein refers to a pathogenic organism that infects a plant.

Percentage of percent identity: “percentage of sequence identity”, as used herein, is determined by comparing two optimally locally aligned sequences over a comparison window defined by the length of the local alignment between the two sequences. The amino acid sequence in the comparison window may comprise additions or deletions (e. g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Local alignment between two sequences only includes segments of each sequence that are deemed to be sufficiently similar according to a criterion that depends on the algorithm used to perform the alignment (e. g. BLAST). The percentage identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Add. APL. Math. 2:482, by the global homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85: 2444, 1988), by heuristic implementations of these algorithms (NCBI BLAST, WU-BLAST, BLAT, SIM, BLASTZ), or by inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. The term “substantial sequence identity” between polynucleotide or polypeptide sequences refers to polynucleotide or polypeptide comprising a sequence that has at least 50% sequence identity, preferably at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using the programs.

Query nucleic acid and amino acid sequences can be searched against subject nucleic acid or amino acid sequences residing in public or proprietary databases. Such searches can be done using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST v 2.18) program. The NCBI BLAST program is available on the internet from the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov/Blast.cgi). Typically the following parameters for NCBI BLAST can be used: Filter options set to “default”, the Comparison Matrix set to “BLOSUM62”, the Gap Costs set to “Existence: 11, Extension: 1”, the Word Size set to 3, the Expect (E threshold) set to 1e-3, and the minimum length of the local alignment set to 50% of the query sequence length. Sequence identity and similarity may also be determined using GenomeQuest™ software (Gene-IT, Worcester Mass. USA).

The term “pesticidal”, as used herein, refers to the ability of a substance or composition to decrease the rate of growth of a pest, i.e., an undesired organism, or to increase the mortality of a pest.

By “suppressive activity” of a biological control agent against a phytopathogen is meant the ability of the agent to suppress, inhibit, stabilize, reverse, slow, or delay the development of the pathogen itself, or the progression of the infection or disease states caused by the pathogen.

Variant: A “variant”, as used herein in reference to a microorganism, is a strain having identifying characteristics of the species to which it belongs, while having at least one nucleotide sequence variation or identifiably different trait with respect to the parental strain, where the trait is genetically based (heritable). For example, for a Microbacterium sp. SGI-SGI-014-006 strain having fungicidal activity, identifiable traits include 1) the ability to suppress the growth of Fusarium graminearum and its teleomorph Gibberella zeae; 2) the ability to suppress the development of head blight disease; 3) having housekeeping genes with greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% sequence identity to the housekeeping genes of Microbacterium sp. SGI-014-006 can be used to confirm a variant as Microbacterium sp. SGI-014-006.

For nucleic acids and polypeptides, the term “variant” is used herein to denote a polypeptide, protein or polynucleotide molecule with some differences, generated synthetically or naturally, in their amino acid or nucleic acid sequences as compared to a reference polypeptide or polynucleotide, respectively. For example, these differences include substitutions, insertions, deletions or any desired combinations of such changes in a reference polypeptide or polypeptide. Polypeptide and protein variants can further consist of changes in charge and/or post-translational modifications (such as glycosylation, methylation, phosphorylation, etc.).

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Methods for Taxonomic Identification

Microorganisms can often be distinguished based on direct microscopic analysis (do all of the cells in a sample look the same on examination), staining characteristics, simple molecular analysis (such as a simply restriction fragment length polymorphism (RFLP) determination), and so forth. In addition to the illustrative examples of such taxonomic analysis techniques as described at Examples 2-3 of the present disclosure, taxonomic identification of a microorganism can involve up to several different levels of analysis, and each analysis can be based on a different characteristic of the organism. Such taxonomic analyses can include nucleic acid-based analysis (e.g., analysis of individual specific genes, either as to their presence or their exact sequence, or expression of a particular gene or a family of genes), protein-based analysis (e.g., at a functional level using direct or indirect enzyme assays, or at a structural level using immuno-detection techniques), and so forth.

a. Nucleic acid-based analysis: One of ordinary skill in the art will appreciate that a wide variety of nucleic acid-based techniques are known and can be useful in obtaining the taxonomic identification of a given microorganism. These techniques can be used to identify cells by gene sequence or to identify cells that have particular genes or gene families. Common gene families useful for taxonomic studies include 16S gene family, actin gene family, and recombinase A (recA) gene family. These methods typically include amplifying and sequencing genes from very small numbers of cells, and therefore often overcome the problems of concentrating cells and their DNA from dilute suspensions. The term “nucleic acid amplification” generally refers to techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen. Techniques useful for nucleic acid amplification are well known in the art. An example of nucleic acid amplification is the polymerase chain reaction (PCR), in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. Other examples of in vitro amplification techniques include strand displacement amplification; transcription-free isothermal amplification; repair chain reaction amplification; ligase chain reaction; gap filling ligase chain reaction amplification; coupled ligase detection and PCR; and RNA transcription-free amplification.

In addition to the illustrative example primers provided herein, see, e.g. Examples 2-3 and the Sequence Listing, primers have also been designed routinely, and new ones are continually being designed, for individual species or phylogenetic groups of microorganisms. Such narrowly targeted primers can be used with the methods described herein to screen and/or identify specifically only the microorganisms of interest.

Methods for preparing and using nucleic acid primers are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). Amplification primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Whitehead Institute for Biomedical Research, Cambridge, Mass.). One of ordinary skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 30 consecutive nucleotides of an rRNA-encoding nucleotide or flanking region thereof will anneal to a target sequence with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers can be selected that comprise at least 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of a target nucleotide sequence such as the 16S rRNA.

Common techniques for the preparation of nucleic acids useful for nucleic acid applications (e.g., PCR) include phenol/chloroform extraction or use of one of the many DNA extraction kits that are available on the market. Another way that DNA can be amplified is by adding cells directly to the nucleic acid amplification reaction mix and relying on the denaturation step of the amplification to lyse the cells and release the DNA.

The product of nucleic acid amplification reactions may be further characterized by one or more of the standard techniques that are well known in the art, including electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing. When in hybridization techniques are used for cell identification purposes, a variety of probe labeling methods can be useful, including fluorescent labeling, radioactive labeling and non-radioactive labeling. When nucleic acid sequencing techniques are used, homology search for the nucleotide sequence of the amplified nucleic acid molecules can be conducted using various databases of known sequences, including but not limited to DDBJ/GenBank/EMBL databases.

b. Protein-based analysis: In addition to analysis of nucleic acids, microorganisms can be taxonomically characterized and identified based on the presence (or absence) of specific proteins directly. Such analysis can be based on the activity of the specified protein, e.g., through an enzyme assay or by the response of a co-cultured organisms, or by the mere presence of the specified protein (which can for instance be determined using immunologic methods, such as in situ immunofluorescent antibody staining).

Enzyme assays: By way of example, fluorescent or chromogenic substrate analogs can be included into the growth media (e.g., microtiter plate cultures), followed by incubation and screening for reaction products, thereby identifying cultures on a basis of their enzymatic activities.

Co-cultivation response: In some embodiments of the present invention, the activity of an enzyme carried by a microbial isolate can be assayed based on the response (or degree of response) of a co-cultured organism (such as a reporter organism).

A variety of methods can also be used for identifying microorganisms selected and isolated from a source environment by binding at least one antibody or antibody-derived molecule to a molecule, or more particularly an epitope of a molecule, of the microorganism.

Anti-microorganism protein antibodies may be produced using standard procedures described in a number of texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). The determination that a particular agent binds substantially only to a protein of the desired microorganism may readily be made by using or adapting routine procedures. One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988)).

Shorter fragments of antibodies (antibody-derived molecules, for instance, FAbs, Fvs, and single-chain Fvs (SCFvs)) can also serve as specific binding agents. Methods of making these fragments are routine.

Detection of antibodies that bind to cells on an array of this invention can be carried out using standard techniques, for instance ELISA assays that provide a detectable signal, for instance a fluorescent or luminescent signal.

Isolated Cultures of the Invention

As described in more details in the Examples section of the present disclosure, Applicants have discovered several agriculturally beneficial novel microorganisms, for example, effective suppressors of head blight disease. Particularly, these novel antagonistic microorganisms are effective for reduction of head blight severity and for inhibition of the growth of Fusarium graminearum, a primary causative agent of head blight disease in wheat. The microbial antagonists were identified from a pool of approximately 5,000 microbial strains obtained from wild plant samples collected from several locations in the United States. Initial selection of antagonistic microorganism was based on the ability of the microorganisms to suppress the development of F. graminearum pathogen and that of its teleomorph Gibberella zeae in an in vitro antagonism assay. Selected microbial antagonists were then bio-assayed in a greenhouse study on wheat seedlings, which involved inoculation of the seedlings with the microbial strains, followed by repeated inoculations of F. graminearum spores, for the ability of the microbial strains to reduce the severity of fungal infestation and for their ability to preserve seed yield. The antagonistic microorganisms selected in this manner were found to be effective in reducing the severity of head blight in greenhouse trials.

Taxonomic analysis further determined that each of the antagonistic microorganisms described in the present disclosure are closely related either the bacterial genus Microbacterium, the bacterial genus Bacillus, the bacterial genus Variovorax, or the fungus genus Mycosphaerella.

The genus Microbacterium, the type genus of the family Microbacteriaceae, is generally considered to accommodate Gram-positive, non-spore forming, rod-shaped bacteria that were originally isolated during early studies on lactic acid-producing bacteria. Members of the genus Microbacterium are originally characterized largely by their marked heat resistance, presence in dairies, and production of small amounts of L(+) lactic acid from glucose. In contrast to other genera of the family Microbacteriaceae in which species are characterized by a coherent peptidoglycan type, species of Microbacterium possess ornithine or lysine either in the inter-peptide bridge or at position 3 of B-type peptidoglycan. In other chemotaxonomic properties, such as isoprenoid quinones (MK-11, MK-12, MK-13), polar lipids, fatty acids and base composition of DNA, members of the genus exhibit the usual range of diversity found in other genera of Microbacteriaceae. Two bacterial genera Microbacterium and Aureobacterium can be united according to some taxonomy studies. To date, members of the genus Microbacterium comprise at least 33 species, which have been isolated from a broad range of habitats, including soil, dairy products, plant galls, insects or clinical specimens. Various aspects of their ecology, phylogeny, taxonomy, culture methods, and long-term preservation conditions have recently been summarized by Evtushenko and Takeuchi (2006). One of skill in the art will readily appreciate that microorganism of the genus Microbacterium can be taxonomically identified largely by any of the taxonomic identification techniques described above, including the chemotaxonomic analysis of the cell wall peptidoglycan and comparative 16S rDNA sequence analysis as described in Evtushenko and Takeuchi (2006) and references cited therein, as well as in those described in Examples 2-3 of the present disclosure. As discussed in detail below, to date several naturally occurring microorganisms have been reported as having antagonistic activity against head blight disease. However, there are no reports prior to the present invention that describe a microorganism of the genus Mycobacterium having such antagonistic activity. Further, prior to the present invention, the present inventors were not aware of any methods or processes of using a bacterial strain of the genus Mycobacterium as biocontrol agent in preventing, inhibiting or treating the development of a causative pathogen of head blight disease.

The genus Bacillus is a genus of Gram-positive/variable, spore-forming, rod-shaped bacteria. Similarly to other genera associated with the early history of microbiology, such as Pseudomonas or Vibrio, nearly 266 species members of the Bacillus genus are found ubiquitously, and it is widely considered to be one of the genera with the largest 16S diversity and environmental diversity. Bacillus species can be obligate aerobes or facultative anaerobes, and test positive for the enzyme catalase. Ubiquitous in nature, Bacillus includes both free-living and pathogenic species. Under stressful environmental conditions, the cells produce oval endospores that can stay dormant for extended periods. These characteristics originally defined the genus, but not all such species are closely related, and many have been moved to other genera. In fact, several studies have tried to reconstruct the phylogeny of the genus. The Bacillus-specific study with the most diversity covered is by Xu and Cote [Intl. J. of Syst. Evol. Microbiol. 53 (3): 695-704; 2003], using 16S and the ITS region, where they divide the genus into 10 groups, which includes the nested genera Paenibacillus, Brevibacillus, Geobacillus, Marinibacillus and Virgibacillus. However, according to more recent studies, the genus Bacillus contains a very large number of nested taxa and especially in both 16S and 23S it is considered to be paraphyletic to Lactobacillales (Lactobacillus, Streptococcus, Staphylococcus, Listeria, etc.), due to Bacillus coahuilensis and others (see, e.g., Yarda et al., Syst. Appl. Microbiol. 31 (4): 241-250, 2008; Yarda et al., Syst. Appl. Microbiol. 33 (6): 291-299, 2010]. One particular clade, formed by B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis and B. weihenstephanensis under current classification standards, should be a single species (within 97% 16S identity), but due to medical reasons, they are considered separate species. In addition to the taxonomy analysis methods described at Examples 2-3 of the present disclosure, the phylogenetic and taxonomic analyses of Bacillus species can be performed by a variety of techniques, including those discussed in details in Xu and Cote, 2003; Yarda et al., 2008; Yarda et al., 2010.

The genus Variovorax was originally created by reclassification of Alcaligenes paradoxus as Variovorax paradoxus (Willems et al., 1991), which is widely consider as the type species of this genus. V. paradoxus has been extensively studied as a model for novel biodegradation agents, as well as microbe/microbe and microbe/plant interactions. Other species include V. dokdonensis, V. soli (Kim et al., 2006), and V. boronicumulans (Miwa et al., 2008). Variovorax species are catabolically very diverse and engage in mutually beneficial interactions with other bacterial species in many biodegradations, and therefore possess ecological importance and high application potential. For example, a soil methanotroph, only when co-cultured together with a V. paradoxus strain, exhibits high affinity for methane (a potent greenhouse gas), and this trait is not usually observed in laboratory cultures. Similarly, a close relative of Variovorax has been found to be the central, non-photosynthetic partner within the phototrophic consortium “Chlorochromatium aggregatum.” Some species of the genus Variovorax also have the ability to interfere with the communication of other bacteria. Some yet other species of the genus Variovorax can intimately interact with other biota (e.g., plants) in various ecosystems. Moreover, some Variovorax species, residing in the area just outside plant roots and/or inside a plant, have been reported for being capable of promoting plant growth via the reduction of ethylene levels, the repression of quorum-sensing-controlled pathogenesis, and the increase of resistance to heavy metals, which greatly benefits phytoremediation. In addition to the taxonomy analysis methods described at Examples 2-3 of the present disclosure, the phylogenetic and taxonomic analyses of Variovorax species can be performed by a variety of techniques, including those discussed in details elsewhere herein.

Mycosphaerella is a very large fungus genus, with over 2,000 species names and at least 500 species associated with more than 40 anamorph genera (especially Cercospora, Pseudocercospora, Septoria, Ramularia, etc.). In addition, several thousand anamorphs lack telomorphs. The genus of Mycosphaerella includes species that are pathogens, saprobes, endophytes, or mutualistic associations. Various aspects of their ecology, phylogeny, taxonomy, culture methods, and long-term preservation conditions have recently been reported (see, e.g. Crous et al., Persoonia, 23:119-146,2009). One of skill in the art will readily appreciate that microorganism of the genus Mycosphaerella can be taxonomically identified by any of the taxonomic techniques described above, or a combination thereof. Most common techniques include comparative sequence analyses using 16S rDNA sequences and the internal transcribed spacer regions as described in, for example, Crous et al., Studies in Mycology, 55:235-253, 2006; Crous et al., 2009, supra; Goodwin et al., Phytopathology 91: 648-658, 2001; and those described in Examples 2-3 of the present disclosure.

Deposit of Biological Material

Purified cultures of microbial strains identified as having suppressive activity against head blight disease were deposited in the Agricultural Research Service Culture Collection located at 1815 N. University Street, Peoria, Ill. 61604, USA (NRRL) in accordance with the Budapest Treaty for the purpose of patent procedure and the regulations thereunder (Budapest Treaty). Accession numbers for these deposits are as follows:

SGI Strain ID Accession No Provisional Taxonomy SGI-005-G08 NRRL_-_(—) Microbacterium sp. SGI-010-H11 NRRL 50471 Mycosphaerella sp. SGI-014-C06 NRRL B-50470 Microbacterium sp. SGI-014-G01 NRRL B-50469 Variovorax sp. SGI-015-F03 NRRL B-50760 Bacillus sp. SGI-015-H06 NRRL B-50761 Bacillus sp.

The microbial strains have been deposited under conditions that ensure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposits represent substantially pure cultures of the deposited strains. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Preferred microorganisms of the present invention have all of the identifying characteristics of the deposited strains and, in particular, the identifying characteristics of being able to suppress the development of head blight disease as described herein, and as being able to suppress the development of Fusarium graminearum pathogen and its teleomorph Gibberella zeae as described herein. In particular, the preferred microorganisms of the present invention refer to the deposited microorganisms as described above, and mutants thereof.

Microbiological Compositions

The microbiological compositions of the present invention that comprise isolated microbial strains or cultures thereof can be in a variety of forms, including, but not limited to, still cultures, whole cultures, stored stocks of cells, mycelium and/or hyphae (particularly glycerol stocks), agar strips, stored agar plugs in glycerol/water, freeze dried stocks, and dried stocks such as lyophilisate or mycelia dried onto filter paper or grain seeds. As defined elsewhere herein, “isolated culture” or grammatical equivalents as used in this disclosure and in the art is understood to mean that the referred to culture is a culture fluid, pellet, scraping, dried sample, lyophilate, or section (for example, hyphae or mycelia); or a support, container, or medium such as a plate, paper, filter, matrix, straw, pipette or pipette tip, fiber, needle, gel, swab, tube, vial, particle, etc. that contains a single type of organism. In the present invention, an isolated culture of a microbial antagonist is a culture fluid or a scraping, pellet, dried preparation, lyophilate, or section of the microorganism, or a support, container, or medium that contains the microorganism, in the absence of other organisms.

The present disclosure further provides compositions that contain at least one isolated microbial strains or cultures thereof of the present invention and a carrier. The carrier may be any one or more of a number of carriers that confer a variety of properties, such as increased stability, wettability, dispersability, etc. Wetting agents such as natural or synthetic surfactants, which can be nonionic or ionic surfactants, or a combination thereof can be included in a composition of the invention. Water-in-oil emulsions can also be used to formulate a composition that includes at least one isolated microorganism of the present invention (see, for example, U.S. Pat. No. 7,485,451, incorporated by reference herein). Suitable formulations that may be prepared include wettable powders, granules, gels, agar strips or pellets, thickeners, and the like, microencapsulated particles, and the like, liquids such as aqueous flowables, aqueous suspensions, water-in-oil emulsions, etc. The formulation may include grain or legume products (e.g., ground grain or beans, broth or flour derived from grain or beans), starch, sugar, or oil. The carrier may be an agricultural carrier. In certain preferred embodiments, the carrier is a seed, and the composition may be applied or coated onto the seed or allowed to saturate the seed.

In some embodiments, the agricultural carrier may be soil or plant growth medium. Other agricultural carriers that may be used include water, fertilizers, plant-based oils, humectants, or combinations thereof. Alternatively, the agricultural carrier may be a solid, such as diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, seed cases, other plant and animal products, or combinations, including granules, pellets, or suspensions. Mixtures of any of the aforementioned ingredients are also contemplated as carriers, such as but not limited to, pesta (flour and kaolin clay), agar or flour-based pellets in loam, sand, or clay, etc. Formulations may include food sources for the cultured organisms, such as barley, rice, or other biological materials such as seed, plant parts, sugar cane bagasse, hulls or stalks from grain processing, ground plant material (e.g. “yard waste”) or wood from building site refuse, sawdust or small fibers from recycling of paper, fabric, or wood. Other suitable formulations will be known to those skilled in the art.

In the liquid form, e.g. solutions or suspensions, the microorganisms may be mixed or suspended in water or in aqueous solutions. Suitable liquid diluents or carriers include water, aqueous solutions, petroleum distillates, or other liquid carriers.

Solid compositions can be prepared by dispersing the antagonist microorganisms in and on an appropriately divided solid carrier, such as peat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceous earth, fuller's earth, pasteurized soil, and the like. When such formulations are used as wettable powders, biologically compatible dispersing agents such as non-ionic, anionic, amphoteric, or cationic dispersing and emulsifying agents can be used.

In a preferred embodiment, the compositions contemplated herein prevent attack by head blight disease upon plants, particularly cereal plants, such as wheat, barley, oat, and corn and, when used in sufficient amounts, to act as microbial antagonists. These compositions, similarly to other biocontrol agents, have a high margin of safety because they typically do not burn or injury the plant.

As described in great detail throughout the present disclosure, control of head blight disease may be effected by application of one or more of the microbiological compositions of the present invention to a host plant or parts of the host plant. The compositions can be applied in an amount effective to reduce the level of head blight relative to that in an untreated control. The active constituents are used in a concentration sufficient to inhibit plant pathogen development of the targeted plant pathogen when applied to the cereal plant. As will be apparent to a skilled person in the art, effective concentrations may vary depending upon such factors as: (a) the type of the plant or agricultural commodity; (b) the physiological condition of the plant or agricultural commodity; (c) the concentration of pathogens affecting the plant or agricultural commodity; (d) the type of disease injury on the plant or agricultural commodity; (e) weather conditions (e.g. temperature, humidity); and (f) the stage of plant disease. According to the invention, typical concentrations are those higher than 1×10² CFU/mL of carrier. Preferred concentrations range from about 1×10⁴ to about 1×10⁹ CFU/mL, such as the concentrations ranging from 1×10⁶ to 1×10⁸ CFU/mL. More preferred concentrations are those of from about 35 to about 150 mg dry microbial mass per gram of carrier (dry formulation) or per milliliter of carrier (liquid composition). In solid formulations, the rate of application should be controlled to result in a comparable number of viable cells per unit area of plant tissue surface as obtained by the aforementioned rates of liquid treatment. Typically, the biological control agents of the present invention are biologically effective when delivered at a concentration in excess of 10⁶ CFU/g (colony forming units per gram), preferably in excess of 10⁷ CFU/g, more preferably 10⁸ CFU/g, and most preferably at 10⁹ CFU/g.

In some embodiments, the amount of one or more of the biological control agents in the microbial compositions of the present invention can vary depending on the final formulation as well as size or type of the plant or seed utilized. Preferably, the one or more biological control agents in the microbial compositions are present in about 2% w/w/ to about 80% w/w of the entire formulation. More preferable, the one or more biological control agents employed in the compositions is about 5% w/w to about 65% w/w and most preferably about 10% w/w to about 60% w/w by weight of the entire formulation.

As it will be appreciated by those skilled in the art, the microbiological compositions of the invention may be applied to the wheat plant or other cereals using a variety of conventional methods such as dusting, coating, injecting, rubbing, rolling, dipping, spraying, or brushing, or any other appropriate technique which does not significantly injure the wheat plant or other cereals to be treated. Particularly preferred is the spray method.

Typically, the compositions of the invention are chemically inert; hence they are compatible with substantially any other constituents of the spray schedule. They may also be used in combination with biologically compatible pesticidal active agents as for example, herbicides, nematicides, fungicides, insecticides, and the like. They can also be used in combination with plant growth affecting substances, such as fertilizers, plant growth regulators, and the like, provided that such compounds or substances are biologically compatible.

When used as pesticides or fungicide in their commercially available formulations and in the use forms, prepared from these formulations, the active microbial antagonists and compositions according to the present invention can furthermore be present in the form of a mixture with synergists. Synergists are compounds by which the activity of the active compositions is increased without it being necessary for the synergist added to be active itself.

When used as pesticides in their commercially available formulations and in the use forms, prepared from these formulations, the active microbial antagonists and compositions according to the invention can furthermore be present in the form of a mixture with inhibitors which reduce the degradation of the active compositions after application in the habitat of the plant, on the surface of parts of plants or in plant tissues.

The active microbial antagonists and compositions according to the invention, as such or in their formulations, can also be used as a mixture with known acaricides, bactericides, fungicides, insecticides, microbicides, nematicides, pesticides, or combinations thereof, for example in order to widen the spectrum of action or to prevent the development of resistances in this way. In many cases, synergistic effects result, i.e. the activity of the mixture can exceed the activity of the individual components. A mixture with other known active compounds, such as fertilizers, growth regulators, safeners and/or semiochemicals is also contemplated.

In a preferred embodiment of the present invention, the compositions may further include at least one chemical or biological pesticide. The amount of at least one chemical or biological pesticide employed in the compositions can vary depending on the final formulation as well as the size of the plant and seed to be treated. Preferably, the at least one chemical or biological pesticide employed is about 0.1% w/w to about 80% w/w based on the entire formulation. More preferably, the at least one chemical or biological pesticide is present in an amount of about 1% w/w to about 60% w/w and most preferably about 10% w/w to about 50% w/w.

A variety of pesticides is apparent to one of skill in the art and may be used. Exemplary chemical pesticides include those in the carbamate, organophosphate, organochlorine, and prethroid classes. Also included are chemical control agents such as, but not limited to, benomyl, borax, captafol, captan, chorothalonil, formulations containing copper; formulations containing dichlone, dicloran, iodine, zinc; fungicides that inhibit ergosterol biosynthesis such as but not limited to blastididin, cymoxanil, fenarimol, flusilazole, folpet, imazalil, ipordione, maneb, manocozeb, metalaxyl,oxycarboxin, myclobutanil, oxytetracycline, PCNB, pentachlorophenol, prochloraz, propiconazole, quinomethionate, sodium aresenite, sodium DNOC, sodium hypochlorite, sodium phenylphenate, streptomycin, sulfur, tebuconazole, terbutrazole, thiabendazolel, thiophanate-methyl, triadimefon, tricyclazole, triforine, validimycin, vinclozolin, zineb, and ziram. A variety of insecticidal compounds can be useful for the compositions of the invention, including but not limited to those cited in U.S. Pat. Appl. No. 20110033432A1.

The microbiological compositions of the present invention preferably include at least one biological pesticide. Exemplary biological pesticides that are suitable for use herein and can be included in a microbiological composition according to the present invention for preventing a plant pathogenic disease include microbes, animals, bacteria, fungi, genetic material, plant, and natural products of living organisms. In these compositions, the microorganism of the present invention is isolated prior to formulation with an additional organism. For example, microbes such as but not limited to species of Ampelomyces, Aureobasidium, Bacillus, Beauveria, Candida, Chaetomium, Cordyceps, Cryptococcus, Dabaryomyces, Erwinia, Exophilia, Gliocladium, Mariannaea, Paecilomyces, Paenibacillus, Pantoea, Pichia, Pseudomonas, Sporobolomyces, Talaromyces, and Trichoderma can be provided in a composition with the antagonistic microorganisms of the present invention, with fungal strains of the Muscodor genus being particularly preferred. Use of the microbiological compositions according to the present invention in combination with the microbial antagonists disclosed in U.S. Pat. No. 7,518,040; U.S. Pat. No. 7,601,346; U.S. Pat. No. 6,312,940 is also particularly preferred.

Examples of fungi that can be combined with microbial antagonists and compositions of the present invention in a composition include, without limitation, Muscodor species, Aschersonia aleyrodis, Beauveria bassiana (“white muscarine”), Beauveria brongniartii, Chladosporium herbarum, Cordyceps clavulata, Cordyceps entomorrhiza, Cordyceps facis, Cordyceps gracilis, Cordyceps melolanthae, Cordyceps militaris, Cordyceps myrmecophila, Cordyceps ravenelii, Cordyceps sinensis, Cordyceps sphecocephala, Cordyceps subsessilis, Cordyceps unilateralis, Cordyceps variabilis, Cordyceps washingtonensis, Culicinomyces clavosporus, Entomophaga grylli, Entomophaga maimaiga, Entomophaga muscae, Entomophaga praxibulli, Entomophthora plutellae, Fusarium lateritium, Hirsutella citriformis, Hirsutella thompsoni, Metarhizium anisopliae (“green muscarine”), Metarhizium flaviride, Muscodor albus, Neozygitesfloridana, Nomuraea rileyi, Paecilomyces farinosus, Paecilomyces fumosoroseus, Pandora neoaphidis, Tolypocladium cylindrosporum, Verticillium lecanii, Zoophthora radicans, and mycorrhizal species such as Laccaria bicolor. Other mycopesticidal species will be apparent to those skilled in the art. 100851 The present invention also provides methods of treating a plant by application of any of a variety of customary formulations in an effective amount to either the soil (ie., in-furrow), a portion of the plant (i.e., drench) or on the seed before planting (i.e., seed coating or dressing). Customary formulations include solutions, emulsifiable concentrate, wettable powders, suspension concentrate, soluble powders, granules, suspension-emulsion concentrate, natural and synthetic materials impregnated with active compound, and very fine control release capsules in polymeric substances. In certain embodiments of the present invention, the biological control compositions are formulated in powders that are available in either a ready-to-use formulation or are mixed together at the time of use. In either embodiment, the powder may be admixed with the soil prior to or at the time of planting. In an alternative embodiment, one or both of either the biological control agent or insect control agent is a liquid formulation that is mixed together at the time of treating. One of ordinary skill in the art understands that an effective amount of the inventive compositions depends on the final formulation of the composition as well as the size of the plant or the size of the seed to be treated.

Depending on the fmal formulation and method of application, one or more suitable additives can also be introduced to the compositions of the present invention. Adhesives such as carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or latexes, such as gum arabic, chitin, polyvinyl alcohol and polyvinyl acetate, as well as natural phospholipids, such as cephalins and lecithins, and synthetic phospholipids, can be added to the present compositions.

In a preferred embodiment, the microbiological compositions are formulated in a single, stable solution, or emulsion, or suspension. For solutions, the active chemical compounds (i.e., the pest control agents) are typically dissolved in solvents before the biological control agent is added. Suitable liquid solvents include petroleum based aromatics, such as xylene, toluene or alkylnaphthalenes, aliphatic hydrocarbons, such as cyclohexane or paraffins, for example petroleum fractions, mineral and vegetable oils, alcohols, such as butanol or glycol as well as their ethers and esters, ketones, such as methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone, strongly polar solvents, such as dimethylformamide and dimethyl sulphoxide. For emulsion or suspension, the liquid medium is water. In one embodiment, the chemical control agent and biological control agent are suspended in separate liquids and mixed at the time of application. In a preferred embodiment of suspension, the insect control agent and biologic are combined in a ready-to-use formulation that exhibits a shelf-life of at least two years. In use, the liquid can be sprayed or can be applied foliarly as an atomized spray or in-furrow at the time of planting the crop. The liquid composition can be introduced to the soil before germination of the seed or directly to the soil in contact with the roots by utilizing a variety of techniques known in the art including, but not limited to, drip irrigation, sprinklers, soil injection or soil drenching.

Optionally, stabilizers and buffers can be added, including alkaline and alkaline earth metal salts and organic acids, such as citric acid and ascorbic acid, inorganic acids, such as hydrochloric acid or sulfuric acid. Biocides can also be added and can include formaldehydes or formaldehyde-releasing agents and derivatives of benzoic acid, such as p-hydroxybenzoic acid.

Seed Coating Formulations

In some particularly preferred embodiments, the biocontrol compositions of the present invention are formulated as a seed treatment. The seed treatment preferably comprises at least one insect control agent and at least one biological control agent. It is contemplated that the seeds can be substantially uniformly coated with one or more layers of the biocontrol compositions disclosed herein using conventional methods of mixing, spraying or a combination thereof through the use of treatment application equipment that is specifically designed and manufactured to accurately, safely, and efficiently apply seed treatment products to seeds. Such equipment uses various types of coating technology such as rotary coaters, drum coaters, fluidized bed techniques, spouted beds, rotary mists or a combination thereof. Liquid seed treatments such as those of the present invention can be applied via either a spinning “atomizer” disk or a spray nozzle which evenly distributes the seed treatment onto the seed as it moves though the spray pattern. Preferably, the seed is then mixed or tumbled for an additional period of time to achieve additional treatment distribution and drying. The seeds can be primed or unprimed before coating with the inventive compositions to increase the uniformity of germination and emergence. In an alternative embodiment, a dry powder formulation can be metered onto the moving seed and allowed to mix until completely distributed.

Another aspect of the invention provides seeds treated with the subject microbiological compositions. One embodiment provides seeds having at least part of the surface area coated with a microbiological composition according to the present invention. In a specific embodiment, the microorganism-treated seeds have a spore concentration or microbial cell concentration from about 10⁶ to about 10⁹ per seed. The seeds may also have more spores or microbial cells per seed, such as, for example 10¹⁰, 10¹¹ or 10¹² spores per seed. The microbial spores and/or cells can be coated freely onto the seeds or, preferably, they can be formulated in a liquid or solid composition before being coated onto the seeds. For example, a solid composition comprising the microorganisms can be prepared by mixing a solid carrier with a suspension of the spores until the solid carriers are impregnated with the spore or cell suspension. This mixture can then be dried to obtain the desired particles.

In some other embodiments, it is contemplated that the solid or liquid biocontrol compositions of the present invention further contain functional agents capable of protecting seeds from the harmful effects of selective herbicides such as activated carbon, nutrients (fertilizers), and other agents capable of improving the germination and quality of the products or a combination thereof.

Seed coating methods and compositions that are known in the art can be particularly useful when they are modified by the addition of one of the embodiments of the present invention. Such coating methods and apparatus for their application are disclosed in, for example, U.S. Pat. Nos. 5,918,413; 5,554,445; 5,389,399; 4,759,945; and 4,465,017. Various seed coating compositions are disclosed, for example, in U.S. Pat. Appl. Nos. US20110033432, US20100154299, U.S. Pat. Nos. 5,939,356; 5,876,739, 5,849,320; 5,791,084, 5,661,103; 5,580,544, 5,328,942; 4,735,015; 4,634,587; 4,372,080, 4,339,456; and 4,245,432, among others.

A variety of additives can be added to the seed treatment formulations comprising the inventive compositions. Binders can be added and include those composed preferably of an adhesive polymer that can be natural or synthetic without phytotoxic effect on the seed to be coated. The binder may be selected from polyvinyl acetates; polyvinyl acetate copolymers; ethylene vinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, including ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses and carboxymethylcellulose; polyvinylpyrolidones; polysaccharides, including starch, modified starch, dextrins, maltodextrins, alginate and chitosans; fats; oils; proteins, including gelatin and zeins; gum arabics; shellacs; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonates; acrylic copolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene.

Any of a variety of colorants may be employed, including organic chromophores classified as nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. Other additives that can be added include trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc. A polymer or other dust control agent can be applied to retain the treatment on the seed surface.

In some specific embodiments, in addition to the microbial cells or spores, the coating can further comprise a layer of adherent. The adherent should be non-toxic, biodegradable, and adhesive. Examples of such materials include, but are not limited to, polyvinyl acetates; polyvinyl acetate copolymers; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, such as methyl celluloses, hydroxymethyl celluloses, and hydroxymethyl propyl celluloses; dextrins; alginates; sugars; molasses; polyvinyl pyrrolidones; polysaccharides; proteins; fats; oils; gum arabics; gelatins; syrups; and starches. More examples can be found in, for example, U.S. Pat. No. 7,213,367 and U.S. Pat. Appln. No. US20100189693.

Various additives, such as adherents, dispersants, surfactants, and nutrient and buffer ingredients, can also be included in the seed treatment formulation. Other conventional seed treatment additives include, but are not limited to, coating agents, wetting agents, buffering agents, and polysaccharides. At least one agriculturally acceptable carrier can be added to the seed treatment formulation such as water, solids or dry powders. The dry powders can be derived from a variety of materials such as calcium carbonate, gypsum, vermiculite, talc, humus, activated charcoal, and various phosphorous compounds.

In some embodiment, the seed coating composition can comprise at least one filler which is an organic or inorganic, natural or synthetic component with which the active components are combined to facilitate its application onto the seed. Preferably, the filler is an inert solid such as clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (for example ammonium salts), natural soil minerals, such as kaolins, clays, talc, lime, quartz, attapulgite, montmorillonite, bentonite or diatomaceous earths, or synthetic minerals, such as silica, alumina or silicates, in particular aluminum or magnesium silicates.

The seed treatment formulation may further include one or more of the following ingredients: other pesticides, including compounds that act only below the ground; fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadixyl, and isomers of each of those materials, and the like; herbicides, including compounds selected from glyphosate, carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils, phenoxys, ureas, and benzoic acids; herbicidal safeners such as benzoxazine, benzhydryl derivatives, N,N-diallyl dichloroacetamide, various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone, naphthalic anhydride compounds, and oxime derivatives; fertilizers; and biocontrol agents such as other naturally-occurring or recombinant bacteria and fungi from the genera Rhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma, Glomus, Gliocladium and mycorrhizal fungi. These ingredients may be added as a separate layer on the seed or alternatively may be added as part of the seed coating composition of the invention.

Preferably, the amount of the novel composition or other ingredients used in the seed treatment should not inhibit germination of the seed, or cause phytotoxic damage to the seed.

The microorganism-treated seeds may also be further enveloped with a film overcoating to protect the coating. Such overcoatings are known in the art and may be applied using fluidized bed and drum film coating techniques.

In principle, any plant seed capable of germinating to form a plant that is susceptible to attack by nematodes and/or pathogenic fungi can be treated in accordance with the invention. Suitable seeds include those of cereals, coffee, cole crops, fiber crops, flowers, fruits, legume, oil crops, trees, tuber crops, vegetables, as well as other plants of the monocotyledonous, and dicotyledonous species. Preferably, crop seeds are coated which include but are not limited to bean, carrot, corn, cotton, grasses, lettuce, peanut, pepper, potato, rapeseed, rice, rye, sorghum, soybean, sugar beet, sunflower, tobacco, and tomato seeds. Most preferably, barley or wheat (spring wheat or winter wheat) seeds are coated with the present compositions.

Also provided, in another aspect of the present invention, is a novel cereal plant created by artificially introducing a microbial endophyte of the invention into a cereal plant that is free of endophytic microorganisms. In some embodiments of this aspect, the microbial endophyte introduced into the cereal plant may be an endophytic antagonist having suppressive activity against head blight disease or a causative agent of head blight disease. Furthermore, the endophytic antagonist introduced into the cereal plant may be the fungal strain SGI-010-H11. A variety of methods previously found effective for the introduction of a microbial endophyte into cereal grass species are known in the art. Examples of such methods include those described in U.S. Pat. Appl. No. 20030195117A1, U.S. Pat. Appl. No. 20010032343A1, and U.S. Pat. No. 7,084,331, among others. It will become apparent to those skilled in the art that many of the aforementioned methods can be useful for the making of a novel cereal plant of the invention.

After artificial infection, it is preferred that DNA of the isolated endophytic antagonist is amplified by PCR and the antagonist is confirmed by carrying out a homology search for the DNA amplified. Further it is preferred that a foreign gene that expresses an identifiable means is introduced into the above-mentioned endophytic antagonist, and the presence of the colonization of the above-mentioned endophytic antagonist infecting the plant is confirmed by the above-identifiable means using the foreign gene.

Preparing the Biocontrol Compositions According to the Present Invention

Cultures of the microbial antagonists may be prepared for use in the biocontrol compositions of the invention using standard static drying and liquid fermentation techniques known in the art. Growth is commonly effected in a bioreactor.

A bioreactor refers to any device or system that supports a biologically active environment. As described herein a bioreactor is a vessel in which microorganisms including the microbial antagonists of the invention can be grown. A bioreactor may be any appropriate shape or size for growing the microorganisms. A bioreactor may range in size and scale from 10 mL to liter's to cubic meters and may be made of stainless steel or any other appropriate material as known and used in the art. The bioreactor may be a batch type bioreactor, a fed batch type or a continuous-type bioreactor (e.g., a continuous stirred reactor). For example, a bioreactor may be a chemostat as known and used in the art of microbiology for growing and harvesting bacteria. A bioreactor may be obtained from any commercial supplier (See also Bioreactor System Design, Asenjo & Merchuk, CRC Press, 1995).

For small scale operations, a batch bioreactor may be used, for example, to test and develop new processes, and for processes that cannot be converted to continuous operations.

Microorganisms grown in a bioreactor may be suspended or immobilized. Growth in the bioreactor is generally under aerobic conditions at suitable temperatures and pH for growth. For the organisms of the invention, cell growth can be achieved at temperatures between 5 and 37° C., with the preferred temperature being in the range of 15 to 30° C., 15 to 28° C., 20 to 30° C., or 15 to 25° C. The pH of the nutrient medium can vary between 4.0 and 9.0, but the preferred operating range is usually slightly acidic to neutral at pH 4.0 to 7.0, or 4.5 to 6.5, or pH 5.0 to 6.0. Typically, maximal cell yield is obtained in 20-72 hours after inoculation.

Optimal conditions for the cultivation of antagonists of this invention will, of course, depend upon the particular strain. However, by virtue of the conditions applied in the selection process and general requirements of most microorganisms, a person of ordinary skill in the art would be able to determine essential nutrients and conditions. The microbial antagonists would typically be grown in aerobic liquid cultures on media which contain sources of carbon, nitrogen, and inorganic salts that can be assimilated by the microorganism and supportive of efficient cell growth. Preferred carbon sources are hexoses such as glucose, but other sources that are readily assimilated such as amino acids, may be substituted. Many inorganic and proteinaceous materials may be used as nitrogen sources in the growth process. Preferred nitrogen sources are amino acids and urea but others include gaseous ammonia, inorganic salts of nitrate and ammonium, vitamins, purines, pyrimidines, yeast extract, beef extract, proteose peptone, soybean meal, hydrolysates of casein, distiller's solubles, and the like. Among the inorganic minerals that can be incorporated into the nutrient medium are the customary salts capable of yielding calcium, zinc, iron, manganese, magnesium, copper, cobalt, potassium, sodium, molybdate, phosphate, sulfate, chloride, borate, and like ions. Without being limited thereto, use of potato dextrose liquid medium for fungal antagonists and R2A broth premix for bacterial strains is preferred.

Throughout this disclosure, various information sources are referred to and incorporated by reference. The information sources include, for example, scientific journal articles, patent documents, textbooks, and World Wide Web browser-inactive page addresses. The reference to such information sources is solely for the purpose of providing an indication of the general state of the art at the time of filing. While the contents and teachings of each and every one of the information sources can be relied on and used by one of skill in the art to make and use embodiments of the invention, any discussion and comment in a specific information source should in no way be considered as an admission that such comment was widely accepted as the general opinion in the field.

The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and embodiments will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.

It should also be understood that the following examples are offered to illustrate, but not limit, the invention.

EXAMPLES Example 1 Discovery of the Antagonistic Microorganisms Capable of Suppressing the Development of Fusarium graminaerum and Gibberella zeae, Causative Agents of Head Blight Disease

This example describes a high-throughput process of collecting and screening candidate microorganisms that has been developed internally at Synthetic Genomics, Inc. to isolate strains of microorganisms having suppressive activity against causative agents of head blight disease, particularly against Fusarium graminearum. Novel microbial antagonistic strains were isolated from plant tissue samples collected from several locations across the United States. The bacterial strains SGI-014-006, SGI-014-G01, SGI-015-F03, and SGI-015-H06 were isolated from plant root tissues collected from Eagle Peak Preserve near Julian, CA. The fungal strain SGI-010-H11 and the bacterial strain SGI-005-G08 were isolated from stem tissues of two different plant samples collected from San Elijo Lagoon, Encinitas, CA.

The microorganisms were isolated as follows. For bacterial strain isolation, plant root tissues were sonicated and subjected to serial dilutions on 2×YT (yeast extract and tryptone) and N-free agar medium plates. Individual colonies were then selected based on morphological characteristics, and individually cultured in liquid broth medium. For fungal isolation, plant tissue was first surface-sterilized by dipping in 70% ethanol and briefly passing through a flame. The tissue was then dissected and placed on potato dextrose agar (PDA) medium, followed by incubation at room temperature. When mycelial growth was observed, a segment of mycelial growth was then transferred to another PDA plate. Strains of microorganisms were isolated, purified and preserved at −80° C. in 15% glycerol at for bacterial strains and on dried barley seed for fungal strains.

Strains of microorganisms isolated as described above were thereafter assayed for their ability to suppress mycelial growth of F. graminearum in an in vitro antagonism test, which was performed on agar plates containing potato dextrose agar (PDA) growth medium according to a high-throughput screening procedure described in U.S. Pat. Appl. No. US20120107915A1, which is incorporated by reference herein, with minor modifications. Briefly, isolated strains of microorganisms were grown on one-fifth strength Tryptic soy broth agar (TSBA/5) for 24 hours prior to use. Conidial inoculum of F. graminearum NRRL-5883 was produced by hyphal tipping an actively growing colony of the fungus and transferring the hyphal strands to PDA agar medium. After incubating the plates for 7 days at 25° C. using a 12 h/day photoperiod, conidia were washed from PDA plates using a weak phosphate buffer (0.004% phosphate buffer, pH 7.2, with 0.019% MgCl₂). A suspension of conidia of F. graminearum in the weak phosphate buffer (1×10⁵ conidia/ml) was then immediately spread over the agar surface, and the plates were then incubated at 25° C. for 48-72 hours. To initiate the antagonism test, cells of isolated microbial strains were point-inoculated at equal distances inside the perimeter of the plate. After five days, the strains were scored as antibiosis positive when a visibly clear area that lacked mycelial growth existed around the perimeter of the microbial colonies.

More than 4,000 microbial strains were isolated and assayed by the procedure described above. Of those, six strains were found to significantly suppress the mycelial growth of F. graminearum NRRL-5883 on PDA medium. These new microbial antagonists are identified as SGI-005-G08, SGI-010-H11, SGI-014-006, SGI-014-G01, SGI-015-F03, and SGI-015-H06.

Antagonism test was also performed for the new microbial antagonists for their ability to suppress the growth of fungal pathogen Gibberella zeae, which is a teleomorphic species of Fusarium graminearum. All procedures were identical to those described above, except that the pathogen strain tested in this assay was a pathogenic strain of the fungus Gibberella zeae. Of these six microbial antagonists, four strains SGI-010-H11, SGI-014-006, SGI-015-F03, and SGI-015-1106 were found to significantly suppress the mycelial growth of Gibberella zeae.

Example 2 DNA Extraction, Sequencing and Taxonom

Fungal Cell Lysis and Acquiring ITS-5.8S rDNA Sequence Information

The fungal biomass was transferred to a 96-well PCR microplate containing 50 μl of a 2× lysis buffer (100 mM Tris HCL, pH 8.0, 2 mM EDTA, pH 8.0, 1% SDS, 400 μg/ml Proteinase K). Lysis conditions were as follows: 55° C. for 60 minutes, 94° C. for 4 minutes. An aliquot of the lysis product was used as the source of template DNA for PCR amplification. The ITS-5.8S rDNA sequence was amplified via PCR using two primers M13-ITS1 (SEQ ID NO: 15) and ITS4 M13-tailed (SEQ ID NO: 16).

For amplification of the ITS-5.8S rDNA region, each PCR mixture was prepared in a 20-μl final volume reaction containing 4 μl from the fungal lysis reaction, 0.2 μM of each primer (ITS1/ITS4), 6% Tween-20, and 10 μl of 2× ImmoMix (Bioline USA Inc, Taunton, Mass.). PCR conditions were as follows: 94° C. for 10 minutes; 94° C. for 30 seconds, 52° C. for 30 seconds, 72° C. for 75 seconds for 30 cycles; 72° C. for 10 minutes. A 2-μl aliquot of the PCR product was run on a 1.0% agarose gel to confirm a single band of the expected size. Positive bands were sent out for Sanger sequencing in the forward and reverse directions using M13 primers. Sequence of the 5.8S intergenic region (ITS) of the fungal strain SGI-010-H11 is provided in the Sequence Listing as SEQ ID NO: 11. Homology search for the determined nucleotide sequence of the 5.8S intergenic region (ITS) was conducted using the DDBJ/GenBank/EMBL database. Subsequently, the phylogenetic relationship of the nucleotide sequence of the 5.8S intergenic region (ITS) was analyzed among the isolated fungal antagonist SGI-010-H11 described herein, microorganisms of the genera and species that exhibit high sequence homologies to the isolated fungal antagonist SGI-010-H11, and other wide varieties of microorganism genera and species, using the ClustalW phylogenetic tree building program. The fungal strain SGI-010-H11 is considered to be related to the family of Mycosphaerellaceae based upon ˜97% similarity of its ITS-5.8S rDNA sequence to those of Mycosphaerella punctiformis (GenBank EU343182) and Ramularia pratensis (GenBank EU019284), whose 5.8S ITS sequences show clear relatedness to Mycosphaerellaceae.

Bacterial Cell Lysis and Acquiring 16S rRNA Sequence Information

A 20-μl aliquot of cell suspension was transferred to a 96-well PCR plate containing 20 μl of a 2× lysis buffer (100 mM Tris HCL, pH 8.0, 2 mM EDTA, pH 8.0, 1% SDS, 400 μg/ml Proteinase K). Lysis conditions were as follows: 55° C. for 30 minutes, 94° C. for 4 minutes. An aliquot of the lysis product was used as the source of template DNA for PCR amplification. The 16S rRNA sequence was amplified via PCR using M13-27F (SEQ ID NO: 17) and 1492R M13-tailed (SEQ ID NO: 18) primers.

For amplification of 16S rRNA region, each PCR mixture was prepared in a 20-μl final volume reaction containing 4 μl from the bacterial lysis reaction, 2 μM of each primer (27F/1492R), 6% Tween-20, and 10 μl of 2× ImmoMix (Bioline USA Inc, Taunton, Mass.). PCR conditions were as follows: 94° C. for 10 minutes; 94° C. for 30 seconds, 52° C. for 30 seconds, 72° C. for 75 seconds for 30 cycles; 72° C. for 10 minutes. A 2-μl aliquot of the PCR product was run on a 1.0% agarose gel to confirm a single band of the expected size. Positive bands were sent out for Sanger sequencing in the forward and reverse directions using M13 primers. Sequences of the 16S rRNA of the bacterial strains SGI-014-006, SGI-005-G08, SGI-014-G01, SGI-015-F03, and SGI-015-H06 are approximately 1.4-Kb in length and are provided in the Sequence Listing as SEQ ID NOs: 1, 10, 12, 13, and 14 respectively. Homology search for the determined nucleotide sequence was conducted using the DDBJ/GenBank/EMBL database. Subsequently, the phylogenetic relationship of the nucleotide sequence of the 16 rRNA genes was analyzed among the isolated bacterial antagonists described herein, bacteria of the genera and species that exhibit high sequence homologies to the isolated bacterial antagonists, and other wide varieties of bacterial genera and species, using the ClustalW phylogenetic tree building program. Sequence identity and similarity were also determined using GenomeQuest™ software (Gene-IT, Worcester Mass. USA). The sequence analysis result revealed that the bacterial isolate SGI-014-G01 can be considered to be related to the genus of Variovorax based upon ˜99% similarity of its 16S rRNA sequence to those of several Variovorax species, including Variovorax sp. R-21938 (GenBank AJ786799) and Variovorax paradoxus (GenBank GU186109), whose 16S rRNA sequences show clear relatedness to Variovorax sp. Further, the sequence analysis result also revealed that the two bacterial isolates SGI-015-F03 and SGI-015-H06 can be considered to be related to the family of Bacillaceae, based upon >99% similarity of their respective 16S sequences to those of several Bacillus spp.

The sequence analysis result revealed that the two bacterial isolates SGI-014-006 and SGI-055-G08 can be considered to be related to the family of Microbacteriaceae based upon >99% similarity of their respective 16S sequences to those of several Microbacterium spp. Notably, the 1430-nt sequence of the 16S rRNA gene of SGI-014-006 (SEQ ID NO: 1) is identical to the 16S rRNA gene of a Microbacterium oxydans strain DSM 20578 and several other Microbacterium sp. strains (e.g., GenBank sequences Y17227.1, FJ200406, EU086800, and EU714335) over its entire 1430-nt length.

In particular, among the thousands of microbial strains that were isolated and tested for the ability to suppress Fusarium development in antagonism assays as described in Example 1, a total of eighty-eight strains was subsequently identified as Microbacterium species based upon the sequence similarity of their 16S sequences to those of known Microbacterium spp. However, Applicants have found that SGI-014-006 and SGI-055-G08 were the only two Microbacterium strains that possessed suppressive activity against Fusarium graminearum. To date, as discussed above, several naturally occurring microbes have been reported as having antagonistic activity against head blight disease. However, there are no reports prior to the present invention that describe a microorganism of the genus Mycobacterium having such antagonistic activity. Further, prior to the present invention, the present inventors were not aware of any methods or processes of using a bacterial strain of the genus Mycobacterium as biocontrol agent in preventing, inhibiting or treating the development of a causative pathogen of head blight disease.

Example 3 Sequence Analysis of Housekeeping Genes from the Isolate SGI-014-C06

A phylogenetic study of several housekeeping genes from 27 species of the genus Microbacterium has been reported recently by Richert et al. (Syst. Appl. Microbiol. 30:102-108, 2007). The study concluded that although the merits of the 16S rRNA sequence analysis for systematic taxonomy are unsurpassed, sole emphasis on a single taxonomic parameter should not guide systematic conclusions. As disclosed above, the nucleotide sequence of the 16S rRNA gene of SGI-014-006 (SEQ ID NO: 1) is identical to the 16S rRNA gene of several Microbacterium sp. strains, including the nucleotide sequence of a 16S rRNA gene of a Microbacterium oxydans strain DSM 20578 that was included in Richert et al. (2007) study (GenBank Accession Y17227.1). Applicants proceeded to perform a phylogenetic analysis on four housekeeping genes of the isolate SGI-014-006, which are DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA), and polyphosphate kinase (ppk). Toward this end, the entire genome of the isolate SGI-014-006 was shot-gun sequenced, assembled and annotated by using procedures described in PCT Patent Application No. WO2010115156A2. Genomic DNA was prepared from a fresh culture of SGI-014-006. Cell pellet was used for high molecular weight DNA extraction using the UltraClean® Mega Soil DNA Isolation Kit (Cat. No 12900-10) from MO BIO Laboratories, Inc. according to the manufacture's recommended protocol. The genomic DNA from SGI-014-006 was then prepared for shotgun 454-pyrosequencing. Genomic DNA (7.5 μg) was used for library construction according to the recommended protocol (454 Life Sciences) for single long reads. The sequences were generated by two GS FLX Titanium series sequencing runs.

The sequences of four housekeeping genes gyrB, rpoB, recA, and ppk of the isolate SGI-014-006 are provided in the Sequence Listing. Homology search for the determined nucleotide sequences was conducted using the DDBJ/GenBank/EMBL database. Sequence identity and similarity were also determined using GenomeQuest™ software (Gene-IT, Worcester Mass. USA). As discussed in details below, the result of sequence analysis of the housekeeping genes revealed that the isolate SGI-014-006 described in the present disclosure is a novel bacterial strain and can be considered to be related to the family of Microbacteriaceae.

The polynucleotide sequence of the gyrB gene of SGI-014-006 has the greatest sequence identity with a gyrB gene of Microbacterium testaceum having GenBank accession number AP012052 (82.69% over a 936/2172 nucleotide alignment). When compared to the gyrB gene of the Microbacterium oxydans strain DSM 20578 (GenBank AM181493; Richert et al., 2007), the sequence homologies between the two genes were ˜62% identical at the nucleotide level and ˜37% identical at the amino acid level.

The polynucleotide sequence of the rpoB gene of SGI-014-006 has the greatest sequence identity with an rpoB gene of Microbacterium maritypicum having GeneBank accession number AM181582 (96.98% over a 1093/3504 nucleotide alignment). When compared to the rpoB gene of the Microbacterium oxydans strain DSM 20578 (GenBank AM181583; Richert et al., 2007), the sequence homologies between the two genes were ˜96% identical at the nucleotide level and 99% identical at the amino acid level.

The polynucleotide sequence of the recA gene of SGI-014-006 has the greatest sequence identity with a recA gene of Microbacterium testaceum having GeneBank accession number AP012052 (85.45% over a 962/1188 nucleotide alignment). When compared to the recA gene of the Microbacterium oxydans strain DSM 20578 (GenBank AM181527; Richert et al., 2007), the sequence homologies between the two genes were ˜92% identical at the nucleotide level and 100% identical at the amino acid level.

The polynucleotide sequence of the ppk gene of SGI-014-006 has the greatest sequence identity with a ppk gene of Microbacterium luteolum having GenBank accession number AM181554 (91.38% over a 1380/2175 nucleotide alignment). When compared to the ppk gene of the Microbacterium oxydans strain DSM 20578 (GenBank AM181556; Richert et al., 2007), the sequence homologies between the two genes were ˜91% identical at the nucleotide level and ˜98% identical at the amino acid level.

Example 4 Protection of Wheat from Fusarium graminearum Infection using the Microbial Antagonists Microbacterium sp. (NRRL B-50470) Mycophaerella sp. (NRRL 50471), and Variovorax sp. (NRRL B-50469)

Microbial strains that were found positive for antibiosis in the antagonism screen described in Example 1 were further evaluated through a plant-based bioassay in which cells of the microbial strains were applied directly onto seeds of a susceptible cereal cultivar, followed by inoculation with conidial spores of F. graminearum. The microbial strains were grown to a sufficient turbidity and diluted in water. Two gram of wheat seeds of a susceptible wheat cultivar (Hank; WestBred LLC, Bozeman, Mont.) were sown in one-liter pots containing pasteurized soil medium. After sowing, 20 mL of the microbial culture dilution was transferred on top of sown seeds. The wheat seeds were allowed to germinate in greenhouse conditions under fluorescent lighting with a 14 hour photoperiod. At the onset of wheat flowering, the wheat heads were challenged by spraying with F. graminearum NRRL 5883 conidial spores. Conidial spores were harvested from five day old PDA plates by pouring water with 0.01% Tween20 on plates and scraping spores into suspension. After spraying spores, wheat plants were transferred to a mist chamber with 100% humidity for three days to allow for infection. The treatments were:

1. Untreated: no microbial or chemical fungicide treatment+Fusarium challenge.

2. Non-infected: no microbial or chemical fungicide treatment, no Fusarium challenge.

3. SGI-010-H11: fungal treatment+Fusarium challenge.

4. SGI-014-G01: bacterial treatment+Fusarium challenge.

5. SGI-014-006: bacterial treatment+Fusarium challenge.

Twenty days after infection, watering was stopped and wheat plants were allowed to dry for three weeks before harvesting. Individual wheat heads were harvested and collected. Disease severity was determined for each head showing head blight disease symptoms. Disease severity was calculated as the number of diseased spikelets divided by the total number of spikelets per heads. The results (TABLE 2) revealed that wheat plants treated with each of the three microbial antagonists tested, SGI-014-006 (NRRL B-50470), SGI-010-H11 (NRRL 50471), and SGI-014-G01 (NRRL B-50469), had significantly lower severity and incidence of Fusarium graminearum infestation when compared to the “untreated” control growing in the same conditions (P<0.05).

The seeds from each head were harvested by hand and the seed mass was weighed. The average seed weight per pot in each of the treatments was calculated. As reported in TABLE 3, each of the microbial antagonists was found to significantly reduce yield loss caused by head blight infestation.

TABLE 2 Efficacy of the microbial antagonists in reducing head blight incidence in wheat. Treatment Percent severity (%) STDEV P value Non-infected 1.30 0.0025 <.0001 Untreated 15.46 0.0364 NA SGI-010-H11 7.42 0.0219 0.0005 SGI-014-G01 6.59 0.0315 0.0001 SGI-014-C06 7.21 0.0225 0.0004

TABLE 3 Efficacy of the microbial antagonists in preserving seed yield in wheat plants having head blight disease. Treatment Seed mass (g) P value Non-infected 0.8438 ± 0.1633 0.0191 Untreated 0.4669 ± 0.1209 NA SGI-010-H11 0.6200 ± 0.1844 0.7025 SGI-014-G01 0.7963 ± 0.2032 0.5277 SGI-014-C06 0.6744 ± 0.0948 0.0551

Example 5 Growth Inhibition of Fusarium graminearum by Microbial Antagonists on Wheat Seedlings

The microbial strains found to possess suppressive activity against F. graminearum in antagonism plate assay were investigated further for their ability to reduce the incidence of head blight in wheat. Seeds of a susceptible wheat cultivar (Hank; WestBred LLC, Bozeman, Mont.) were sown in 1-Liter pots each containing pasteurized soil medium in 20 cm diameter plastic pots. Microbial cell suspensions were prepared as follows.2YT, or similar growth medium, broth cultures were inoculated from isolate glycerol stocks or streak plates. Typically, cultures were initiated 48-72 hours prior to use in the growth chamber, greenhouse or field allowing the culture to reach late exponential phase. Isolates that have longer doubling times were initiated further in advance. Cultures were incubated at 30° C. on a rotary shaker at 200 rpm. After growth, the cells were pelleted at 10,000×g for 15 min at 4° C. and re-suspended in 10 mM MgSO₄ buffer (pH 7.0). Cell densities were normalized for each isolate on a CFU/mL basis (cell performing units per milliliter). Typically, ˜10⁹ CFU/mL suspensions were prepared for each isolate and transported to the inoculation site on ice. Inoculations were performed by diluting these cell suspensions 1/20 in irrigation water to a final density 5×10⁷ CFU/mL. For 1-L pot trials, 20 mL of this dilute cell suspension was distributed evenly over the surface of each replicate pot. For micro-trial pots, cell suspensions were not diluted and 1 mL of each 10⁹ CFU/mL suspension was pipetted directly onto the surface of each replicate pot as soil drench on top of the submerged seeds. The seeds and plants were maintained in a greenhouse at room temperature with a 14 h photoperiod of light.

Mycelium of the Fusarium graminearum strain NRRL-5883 was grown on PDA plates for 5 days under constant light. Hyphae and conidia were harvested by pouring a few mL of sterile water (0.01% Tween 20) on the plates and scraping the agar surface with a sterile spatula. The concentration of spores in the inoculum was approximately 2×10⁵ spores/mL, but hyphal fragment concentration was not determined.

Inoculation with F. graminearum began after shoot emerging from the seeds and continued every other evening for 10 days. The inoculum of F. graminearum was applied with a sprayer at about 30 mL per pot. Immediately after each inoculation, the plants were misted with overhead mister. When chemical fungicide was used, the fungicide (Banner Maxx, Syngenta) was prepared by diluting 2 mL of fungicide stock in 1 L of distilled water, and was applied with a sprayer at about 30 mL per pot.

Head blight was evaluated by the severity of Fusarium infestation, as determined by the number of Fusarium colony-forming units per gram (CFU/g) of plant tissues. All treatments were performed in four blocks with four replicate pots. The treatments were:

1. Untreated: no microbial or chemical fungicide treatment, +Fusarium challenge

2. Non-infected: no microbial or chemical fungicide treatment, no Fusarium challenge.

3. Fungicide: chemical fungicide treatment+Fusarium challenge.

4. SGI-010-H11: fungal treatment+Fusarium challenge.

5. SGI-014-G01: bacterial treatment+Fusarium challenge.

6. SGI-014-006: bacterial treatment+Fusarium challenge.

The results, as reported in TABLE 4, revealed that all three microbial treatments provided a significant decrease in FHB severity as compared to the infected control. In particular, two microbial antagonists, SGI-014-006 and SGI-010-H11, significantly reduced the infestation of wheat plants by Fusarium graminearum when compared to the untreated control grown in the same conditions. The protection of wheat plants from Fusarium infestation effected by each of the two microorganisms SGI-014-006 and SGI-010-H11 was statistically comparable to the protection effected by the commercial chemical fungicide. When the microorganism SGI-014-G01 was applied, the observed protection of wheat plants from Fusarium infestation was much less conspicuous and its effectiveness varied greatly among the replicate pots.

TABLE 4 Effect of microbial antagonists on Fusarium infestation. Treatment CFU/g P value Untreated 1487 ± 600  N/A Non-infected 0 ± 0 <.0001 Chemical fungicide 290 ± 146 <.0001 SGI-010-H11 671 ± 450 0.0077 SGI-014-G01 1246 ± 465  0.8387 SGI-014-C06 367 ± 234 0.0001

Example 6 Growth and Storage of the Microbial Antagonists

Mycosphaerella sp.: Several methods were used to store the isolated fungus as a pure culture, one of which was the filter paper technique. The fungus was also allowed to grow on PDA, and then it was cut into small squares which were placed into vials containing 15% glycerol and stored at −70° C. The fungus was also stored at 4° C. by a similar method, using distilled water rather than glycerol. However, one of the preferred methods of storage was on infested sterile barley seed at −80° C.

Bacillus sp., Microbacterium sp. and Variovorax sp.: The isolated bacteria were stored as a pure culture. A bacterial colony was transferred to a vial containing R2A broth liquid medium (Tecknova) and allowed to grow at 30° C. with shaking at 250 rpm for two days. The culture was then transferred into vials containing 15% glycerol and stored at −80° C.

Example 7 Spore Production and Seed Coating Treatments

Spore production: In a typical spore production procedure, one liter of 2× YT growth medium (16 g/L Tryptone, 10 g/L yeast extract, 5 g/L NaCl) was inoculated with 5 mL starter culture or petri dish scraping and incubated overnight at 30° C. in a rotating shaker that was set at 225 rpm. Bacterial cells were pelleted by centrifugation, and washed lx with PBS buffer (8 g/L NaCl; 0.2 g/L KCl; 1.44 g/L Na₂HPO₄; 0.24 g of KH₂PO₄; pH 7.4). Cells were resuspended in CDSM medium (Hageman et al., J. Bacteriol., 438-441, 1984), and were grown for four additional nights at 30° C. in the rotating shaker. Sporulation in the bacterial culture was monitored daily by using phase contrast microscope until the entire culture was virtually made up of free-floating spores. The incubation time typically took less than four days or more than six days depending on the species. Under phase contrast microscope, endospores were detected within cells as bright white oblate spheroids. Bacterial spores were pelleted by centrifugation at 10,000×g for 15 minutes, washed two times with sterile dH₂O and, if necessary, concentrated down to 50 mL and could either be used immediately or refrigerated for later use. Spore concentration was measured at OD₆₀₀. This procedure typically produced at least 2000 OD's of bacterial spores.

In particular, several bacterial strains of the present invention, e.g. Bacillus sp. SGI-015-F03, could conveniently produce large amounts of spores after 4 days of incubation with a simple inoculation of a large overnight starter culture (˜15 mL) in to 1 liter of 2× SG growth medium, followed by a 4-day incubation at appropriate temperature in a rotating shaker set at 225 rpm. The recipe of the 2× SG growth medium was as follows: 16 g/L Nutrient Broth; 0.25 g/L MgSO₄; 2.0 g/L KCl; 0.15 g/L CaCl₂.2H₂O; 0.025 g/L MnCl₂.2H₂O; 0.28 mg/L FeSO₄.7H₂O; 1.0 g/L Dextrose.

Seed coating treatments of wheat and corn seeds: Small-scale seed treatment experiments were conducted by following a procedure described in Sudisha et al., 2009 with minor modifications. Typically, a biopolymer stock solution was made by adding 6 gram of gum Arabic powder (MP Biomedical) to 36 mL water in a 50 mL Falcon tube, which was subsequently mixed to homogeneity by using a wheel mixer. A stir plate was used for mixing when larger quantities of coated seeds were needed.

When vegetative cells were used, turbid cultures of actively growing microbial cells were washed with PBS and adjusted to an OD₆₀₀ of ˜5.0. Alternatively, microbial spore suspensions were prepared as described above. Bacterial spores and/or vegetative cells were thoroughly resuspended in ˜32 mL gum Arabic biopolymer stock solution prepared as described above, and resulting suspension was mixed thoroughly in a 1 L bottle. Approximately 400 g of seeds (either wheat or corn) were added to the bottle and vigorously shaken or vortexed to ensure a uniform distribution of the gum/cell suspension. Coated seeds were then spread across a sterile plastic weigh boats to dry in a laminar flow hood until no longer tacky, generally 3-6 hours with periodic mixing. In some instances, seeds coated with spores could be dried overnight. However, seeds coated with vegetative cultures were typically stored away before they completely desiccated. Viability test performed periodically on the microbes used in seed coating formulation showed that the microbes remained viable for at least three months. Germination rate of the coated seeds was determined to be essentially identical to control uncoated seeds.

Example 8 Effect of Microbial Seed Treatments on the Development of Fusarium Head Blight in Wheat

Seeds of a FHB-susceptible wheat cultivar (RB07) were coated with each of the microorganisms SGI-014-006; SGI-015-F03, and SGI-015-H06, in accordance with the procedure described in Example 8. Coated seeds were subsequently sown in one-liter pots containing pasteurized soil medium [(Metromix-Mix 200; Scotts-Sierra Horticultural Products, Marysville, Ohio; and 3 gram of fertilizer 14-14-14 (N-P-K)]. Pots were seeded at 7-8 plants per pot and subsequently thinned to 5 plants per pot at the 3-leaf stage. Pots were arranged in randomized blocks with 5 pots per treatments. The coated seeds were then allowed to germinate in greenhouse conditions under fluorescent lighting with 16 hours of lighting per day. At the onset of wheat flowering (anthesis), the wheat heads were challenged by spraying with F. graminearum NRRL 5883 macroconidial spores at a concentration of 100,000 spores/mL. After spraying spores, wheat plants were transferred to a dew chamber with 100% humidity for three days to allow for infection. The treatments were:

1. Untreated: no microbial or chemical fungicide treatment+Fusarium challenge.

2. SGI-014-006: bacterial seed treatment+Fusarium challenge.

3. SGI-015-F03: bacterial seed treatment+Fusarium challenge.

4. SGI-015-H06: bacterial seed treatment+Fusarium challenge.

Severity of head blight disease was assessed visually at ten days and twenty-one days after infection. Disease severity was determined for each spike showing head blight disease symptoms, and calculated as the percentage of symptomatic spikelets; i.e. the number of spikelets displaying FHB symptoms divided by the total number of spikelets. The results (TABLE 5) revealed that wheat seeds coated with each of the three microbial antagonists tested, SGI-014-006; SGI-015-F03, and SGI-015-H06, had significantly lower severity of Fusarium graminearum infestation when compared to the “untreated” control growing in the same conditions (P<0.05).

TABLE 5 Development of head blight symptoms in wheat following seed treatments with antagonistic microorganisms Infection severity (%) Treatment 10 days post-infection 21 days post infection Untreated control 49.31 66.10 SGI-014-C06 2.08 7.74 SGI-015-F03 23.12 31.53 SGI-015-H06 9.42 19.81

Example 9 Biocontrol of Head-Blight Disease of Wheat in Greenhouse Trials

Each of the microorganisms SGI-014-006; SGI-015-F03, and SGI-015-H06 were further tested at larger-scale greenhouse trials. The trials were conducted in a “plant growth containment room” (PGCR) located at Synthetic Genomics, Inc., and included the following control treatments: infected control, non-infected control, as well as four commercial fungicide benchmarks. The benchmarking chemical fungicides were Banner MAXX® (Syngenta) at a concentration of 2 ml/L and Prosaro® (Bayer CropScience) at a concentration of 3 ml/L, each applied as a foliar spray, following manufacturers' recommendation. The benchmarking biological fungicides were Actinovate® (Natural Industries) and RhizoVital® (ABiTEP GmbH), each applied as a soil drench as per instruction manuals recommended by manufacturers.

The microbial treatments were prepared and applied either as a soil drench or as a seed coat. When microbial treatments were applied as a soil drench, cell suspensions of each of the microorganisms were individually applied to the seeds before covering the seeds with more growth medium. For this purpose, freshly prepared cultures were pelleted to remove growth media and resuspended in 10 mM magnesium sulfate (MgSO₄) buffer. Cell suspensions were then added by pipetting and distributing 20 milliliters evenly across the seeds at a total cell number of ˜10⁹ cells per pot. When microbial treatments were applied as a seed coating, microorganism cells/spores were essentially prepared as described in Example 8 above. Coated seeds were produced by incorporating cell suspensions with a gum Arabic solution to form a tacky coating mixture that was subsequently applied to the seeds. The microbial seed coating was then allowed to dry to form a hard but water soluble biopolymer entrapping the microorganism cells/spores. The goal was to reach a cell titer in the range of at least 10⁶-10⁷ viable cells/seed. In this greenhouse trial experiment, the seed coatings were prepared the day prior to the sowing of the seeds.

Each treatment had four replicates that were positioned in a Randomized Complete Block Design (RCBD) on three leveled racks arranged in two rows along the sides of a growth room. The racks were equipped with fluorescent lights (Agrosun, 5850K) that generated on average 800 μmole of light intensity as measured at the pot surface. Each replicate consisted of four 1-liter pots contained in a half flat. The pots were filled with a synthetic growth medium consisted of a 3:1 ratio (v/v) of Arabidopsis Growth Medium AIS (Lehle Seeds) to sand (Washed Coarse). In each pot, two grams of spring wheat seeds (Hank, WestBred) were scattered on the surface of the growth medium. The pots were bottom watered by keeping ˜2 cm water at their base and observed for germination and growth.

At flowering stage (Feekes 10.5.1) the wheat heads were infected by spraying with a Fusarium graminearum conidial spore suspension. For this greenhouse trial experiment, fungal spores were prepared by plating homogenized mycelia of F. graminearum strain NRRL 5883 onto large PDA plates, followed by five-day incubation under constant light at room temperature. The fungal spores were then harvested by flooding the plates with magnesium sulfate buffer (10 mM MgSO₄; 0.01% Tween 20) and gently scraping the agar surface with a sterile spatula. The spore concentration was adjusted and approximately 50,000 conidial spores were applied per head with a pressurized hand sprayer. The growth room relative humidity was increased to 90% for three days allowing for infection before again being normalized. After wheat seeds had set, watering was stopped and the wheat heads were allowed to dry completely. Individual wheat heads were harvested and collected. Disease severity was determined for each wheat head showing head blight disease symptoms Disease severity was calculated as the number of diseased spikelets divided by the total number of spikelets per head. The results (TABLE 6) revealed that wheat treated with each of the three microbial antagonists tested, SGI-014-006; SGI-015-F03, and SGI-015-H06, had significantly lower severity and incidence of Fusarium graminearum infestation when compared to the “untreated” control growing in the same conditions (P<0.05).

TABLE 6 Efficacy of the microbial antagonists in reducing head blight incidence in wheat. Treatment Infection severity (%) Infection protection % Non-infected  2.1 ± 2.9 N/A Infected 37.2 ± 9.8 0.00 Prosario ®  4.6 ± 2.8 93.00 Banner-MAXX ® 12.5 ± 5.8 70.00 Actinovate ®  38.9 ± 12.1 −5.00 RhizoVital ® 28.0 ± 5.4 26.00 SGI-014-C06 18.4 ± 9.1 30.00 SGI-015-F03 25.4 ± 6.9 54.00 SGI-015-H06 26.6 ± 7.4 34.00

To determine seed yield, wheat heads were individually threshed by hand, the seeds collected and cleaned of chaff and other detritus, counted, and weighed. The total seed yield was determined as the total seed mass produced by 16 pots of each of the treatments. As reported in TABLE 7 each of the three microbial antagonists tested was found to significantly protect the seed yield; i.e. the yield loss caused by head blight infestation was significantly reduced.

TABLE 7 Efficacy of the microbial antagonists in preserving seed yield in wheat plants having head blight disease. Treatment Total seed yield (g) Yield protection (%) Non-infected 14.36 N/A Untreated 11.05 0.00 Prosario ® 13.44 72.00 Banner-MAXX ® 14.41 102.00 Actinovate ® 11.99 28.00 RhizoVital ® 12.65 48.00 SGI-014-C06 14.70 110.00 SGI-015-F03 15.03 120.00 SGI-015-H06 14.14 93.00

Thus, treatments of wheat plants with each of the tested microorganisms significantly preserved the wheat plants against yield loss caused by Fusarium infestation. In particular, wheat plants treated with either SGI-014-006 or SGI-015-F03 showed a significant improvement in total seed yield; i.e. 110% and 120% respectively, when compared to non-infected control. In contrast, the benchmarking fungicides Actinovate®, RhizoVital®, and Prosario® did not appear to significantly protect the treated wheat plants from yield loss caused by Fusarium head blight infestation.

Example 10 Biocontrol of Wheat Scab Under Field Conditions

Microorganism antagonists that were found having suppressive activity against the pathogen F. graminearum and head blight disease in in vitro antagonism assays and greenhouse studies, as described in Examples 4-9, are further evaluated in a series field experiments at different geographical locations throughout the United States. Some experiments are carried out in agricultural areas which are used for microbiological control of wheat scab, where natural disease infection occurs. The wheat variety used in the test is a F. graminearum susceptible variety. Generally, wheat plants with each treatment were sown in plots of 12 rows, about 3-meters long. The space between rows is about 20 cm. Treated plots in each experiment are arranged in a randomized block design. The efficacy of various wettable powder compositions containing the microbial antagonists of the present invention in reducing head blight severity and incidence are also assessed. In these experiments, the microbial antagonists are evaluated either individually or in combination.

In these tests, microorganism antagonists are evaluated in various seed-coating treatments and/or field tests where microbial suspensions are applied directly onto flowering wheat heads. In each of the experiments, the microorganisms are assessed in replicated plots. Antagonist, pathogen, and plant production methods as described in Examples 4-9 can be used in these experiments.

Seed-coating treatment—In addition to the seed coating method described in Example 7 above, a variety of techniques and seed-coating formulations known in the art can be deployed for the treatment of wheat seeds with microbial antagonists, such as those described previously by Fernando et al., 2002; Bello et al., 2002; and Kim et al., 1997. In general, wheat seeds are pre-moistened and stored at room temperature to promote germination. Germinated seeds can be then immersed into microbial suspensions before seeding. The pathogen spores can be inoculated either before or after bacterial inoculation. Antagonist, pathogen, and plant production methods as described in Examples 4 and 5 can be used for these seed treatments.

Disease severity and yield assessment—The antagonists are subjected to further evaluation in greenhouse and field evaluation for their suppressive activity against head blight disease on wheat and barley at flowering stages, by using a variety of methods and procedures, typically those described in, e.g., Schisler, Plant Disease, Vol 86(12), 1350-1356, 2002; Schisler et al. Biological Control, 39:497-506, 2006; and Khan and Doohan, Biological Control, 48:42-47, 2009. In one of such experiments, inoculums of G. zeae are prepared on sterile, yellow dent corn as described by Khan et al. (Biological Control, 29:245-255, 2004). Fully colonized lipid cultures (48-hour culture) of microbial strains are diluted to one-quarter strength with phosphate buffer. Final CFU per mL counts for antagonist treatments are between 1×10⁹ CFU/mL and 6×10⁹ CFU/mL. Treatment suspensions are then applied to flowering wheat heads using a CO₂ backpack sprayer. Treatments are typically applied after sunset to minimize potential UV degradation of antagonist cells. There are 5 replicates per treatment which are arranged in a randomized block design. The primary control treatment consists of plants treated with a buffer solution. A second control consists of untreated plants. Field assessments of head blight disease and incidence are made by evaluating 60 heads per replicate (i.e. 300 heads/treatment) when plant development reaches between mid-milk and soft dough development. Wheat heads are harvested by hand and threshed using an Almaco single plant and head thresher (Almaco, Iowa) when grains reach full maturity. Grain samples obtained from each replicate row are evaluated for 100-kernel weight. Disease severity, incidence, and 100-kernel weight data are analyzed using one-way analysis of variance (ANOVA).

The compositions containing the microbial antagonists of the invention, either individually or in combination, are found to significantly diminish the disease incidence. These results show that the biological control measures using these active microbial agents can play an important role in the management of scab on cereal plants such as wheat plants.

Example 11 Development of Non-Naturally Occurring Cultivars and Breeding Program

Endophytic microorganisms of the present invention are introduced into crop plants, including cereals, of varying genotypes and geographic origin, lacking such endophytic microorganisms, to create plant-endophyte combinations with improved agronomic characteristics, using procedures analogous to those known in the art, including those described in U.S. Pat. Appl. No. 20030195117A1; U.S. Pat. Appl. No. 20010032343A1; and U.S. Pat. No. 7,084,331, among others. Thus, given synthetic plant-endophyte combinations may be created and selected in a breeding/cultivar development program based on their ability to form and maintain a mutualistic combination that results in an agronomic benefit. Rating of agronomic characteristics of the combination may also be utilized in such a breeding program. These characteristics may include, without limitation, drought tolerance, biomass accumulation, resistance to insect infestation, palatability to livestock (e.g. herbivores), ease of reproduction, and seed yield, among others. Such combinations may differ in levels of accumulation of microbial metabolites that are toxic to pests and weeds, including ergot alkaloid levels, loline levels, peramine levels, or lolitrem levels, while displaying desired agronomic characteristics of crop plants, including resistance to insect feeding or infestation, resistance to abiotic stress, palatability to livestock, biomass accumulation, ease of reproduction, and seed yield, among other traits.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that elements of the embodiments described herein can be combined to make additional embodiments and various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments, alternatives and equivalents are within the scope of the invention as described and claimed herein.

Headings within the application are solely for the convenience of the reader, and do not limit in any way the scope of the invention or its embodiments.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically can individually indicated to be incorporated by reference. 

1-23. (canceled)
 24. A synthetic combination comprising a purified bacterial population in association with a seed of a crop plant, wherein the purified bacterial population has a 16S rRNA nucleic acid sequence that is at least 97% identical to a 16S rRNA nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 10, 12, 13, and
 14. 25. An isolated strain comprising a purified bacterial population in association with a seed of a crop plant, wherein the purified microbial population has a 16S rRNA nucleic acid sequence that is at least 97% identical to a 16S rRNA nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 10, 12, 13, and
 14. 